Disordered renal epithelial ion transport results in hypertension and hyperkalemia, which confer increased risk of cardiovascular complications and death. Data from humans and mouse models indicate that inhibition of with-no-lysine (WNK) kinases in transporting renal epithelia has the potential to ameliorate hypertension and hyperkalemia. Recent work has shown that in vitro, Cl- binds to the WNK active site and inhibits its activation. What is unknown, however, is whether Cl- regulates WNK-dependent transepithelial ion transport. To understand this, the ability to measure and manipulate Cl- in a WNK-regulated epithelium is required. However, this is difficult to achieve in mammalian renal tubules. The applicant's long-term goal is to better understand disease-relevant epithelial ion transport mechanisms by using the Drosophila melanogaster renal tubule. This innovative approach harnesses the ease of genetic manipulation in a physiologically accessible transporting epithelium. The overall objective of this proposal is to develop methods to measure and manipulate intracellular Cl- concentrations within the fly renal tubule. The rationale is to enable a future phase of the project, studying whether and how intracellular Cl- regulates transepithelial ion transport through regulation of WNK signaling. The applicant has previously demonstrated that hypotonicity stimulates WNK-dependent transepithelial ion transport in the fly renal tubule. In other renal epithelia, there is a two-phase decrease in intracellular Cl- after hypotonicity-induced swelling. First, there is a dilutional decrease in intracellular Cl-. This is followed by a further decrease in Cl- as cells undergo compensatory regulatory volume decrease mediated by efflux of KCl. Efflux occurs through potassium-chloride cotransporters (KCCs), and/or parallel K+ and Cl- efflux through channels. In Drosophila cultured cells, bestrophin-1 is the swell-activated Cl- channel. The hypothesis here is that inhibition of bestrophin-1 under conditions of hypotonicity-induced swelling will blunt the lowering of intracellular Cl-, while expression of constitutively active KCC will lower intracellular Cl- in isotonic conditions, in the absence of cellular swelling. This hypothesis will be tested in three specific aims: In aim 1, the applicant will determine the optimal means for measuring intracellular Cl- in the Drosophila renal tubule, testing the feasibility of using a transgenic ratiometric fluorescent Cl- sensor. Aim 2 will determine whether bestrophin-1 mediates swell-induced Cl- efflux from tubule epithelial cells by measuring intracellular Cl- in isotonic and hypotonic conditions, with or without bestrophin-1 inhibition by knockdown or dominant-negative transgene expression. In aim 3, mutations will be introduced into predicted phospho-regulatory serines and threonines to generate a constitutively active KCC. Intracellular Cl- will be measured in isotonic (non-swell) conditions. The proposed research is significant, because it will facilitate development of drugs targeting WNK-dependent epithelial ion transport processes, with beneficial effects on hypertension and hyperkalemia, that have decreased risk of off- target effects due to the unique mechanism of kinase regulation by intracellular Cl-.